The aim of this research program is to understand in molecular detail the mechanism by which the replication of the herpes simplex virus type 1 genome is initiated and sustained. Our current model is that following circularization of the linear viral genome, theta type replication is initiated at one or more of the three HSV-1 origins, followed by a switch to rolling circle replication. We propose to reconstitute in vitro with purified enzymes whose structure and mechanism we will examine in detail, both the theta and rolling circle phases of HSV-1 DNA replication. We anticipate that these studies will provide us with an insight into the replication of a significant class of human pathogens. The investigation will be organized along the following lines: A. Structure and mechanism of HSV-1 encoded replication enzymes. 1. Origin binding protein (UL9 protein) a. Unwinding of oris by UL9 protein b. Determination of stoichiometry of binding of UL9 protein to Oris c. Interaction of UL9 protein with cellular DNA polymerase alpha-primase d. Determination of three- dimensional structure of UL9 protein 2. DNA polymerase-UL42 protein complex a. Pre-steady state kinetic analysis of enhancement of processivity by UL42 protein b. Effect of phosphorylation of UL42 protein on processivity enhancement 3. Helicase-primase (Primosome) a. Role of UL8 subunit of primosome in its helicase activity b. Analysis of helicase activity c. Analysis of primase activity B. Reconstitution of Oris-dependent DNA replication 1. General approach 2. Examination of specific conditions 3. Transcriptional Activation of transcription C. Further studies of rolling circle DNA replication by a complex of HSV-1 encoded replication enzymes 1. Assembly of complex 2. Mechanism of switch from theta to rolling circle DNA replication.